Tibaru17

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Tibaru17

  1. 1. Hermi Indita/Endang Retnowati PEMERIKSAAN LIMFOSIT T CD4 + DENGAN FLOW CYTOMETRY
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  6. 6. Aplikasi Klinis Flow Cytometry <ul><li>Monitoring perubahan populasi sel pada penyakit infeksius </li></ul><ul><ul><li>misalnya Limfosit T CD4 + pada HIV </li></ul></ul><ul><li>Identifikasi populasi sel abnormal pada keganasan </li></ul><ul><ul><li>misalnya pada lekemia </li></ul></ul>
  7. 7. Flow Cytometer BD FACSCalibur™ System
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  9. 9. <ul><li>Terdiri dari sheath fluid dan central channel </li></ul><ul><li>Tenaga hidrodinamik sel satu persatu melewati central channel </li></ul>Excitation Optics (laser) Fluidics–Hydrodynamic Focusing Sistem Fluida
  10. 10. Sistem Optik <ul><li>Laser eksitasi fluorokrom yang menempel pada sel </li></ul><ul><li>Filter optik membedakan berbagai macam fluorokrom </li></ul><ul><li>Sinar pencar & emisi fluoresen informasi struktur sel : </li></ul><ul><ul><li>Forward Scatter Channel (FSC): </li></ul></ul><ul><ul><li>Mengukur luas permukaan sel </li></ul></ul><ul><ul><li>Side Scatter Channel (SSC): </li></ul></ul><ul><ul><li>Mendeteksi sinar pada sudut 90 0 </li></ul></ul><ul><ul><li>Mengukur kompleksitas & granulasitas sel </li></ul></ul><ul><li>Detektor yang dipakai : </li></ul><ul><ul><li>Photomultiplier Tubes (PMT) : SSC dan Fluoresen </li></ul></ul><ul><ul><li>Photodiode : FSC </li></ul></ul>
  11. 11. BD FACSCalibur Optics
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  13. 13. <ul><li>Pengumpulan dan analisis data : </li></ul><ul><ul><li>mengubah sinyal analog sinyal digital </li></ul></ul><ul><ul><li>analisa tegangan pulsa : tinggi, luas, lebar </li></ul></ul>Sistem komputer
  14. 14. Prosedur Pemeriksaan Limfosit CD4 + Dengan Menggunakan Flow Sitometer BD FACSCalibur™ System <ul><li>Prinsip : Tabung reagen mengandung antibodi yang ditandai fluorochrome dalam jumlah tertentu. Jika ditambahkan sampel, antibodi tsb akan terikat dengan antigen pd permukaan limfosit dan akan berfluoresensi bila terkena sinar laser. Fluoresensi yang terjadi sebanding dgn jumlah sel yang ada </li></ul>
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  16. 16. Persiapan reagen
  17. 17. Cara kerja
  18. 18. Interpretasi Hasil <ul><li>Nilai normal : </li></ul><ul><ul><li>Dewasa : </li></ul></ul><ul><ul><ul><li>Limfosit T CD4 absolut :≥ 500 / cmm 3 </li></ul></ul></ul><ul><ul><ul><li>Limfosit T CD4 % : ≥ 25% </li></ul></ul></ul><ul><ul><li>Bayi ≥ 12 bulan : </li></ul></ul><ul><ul><ul><li>Limfosit T CD4 absolut : ≥1500/cmm 3 </li></ul></ul></ul><ul><ul><ul><li>Limfosit T CD4% :≥25% </li></ul></ul></ul><ul><ul><li>1 – 5 tahun : </li></ul></ul><ul><ul><ul><li>Limfosit T CD4 absolut : ≥1000/cmm 3 </li></ul></ul></ul><ul><ul><ul><li>Limfosit T CD4 % :≥25% </li></ul></ul></ul>
  19. 19. <ul><li>Nilai absolut dan prosentase limfosit T CD4 + yang sangat menurun </li></ul>Contoh printout pemeriksaan limfosit T CD4 + menggunakan Flow Cytometer BD FACSCalibur
  20. 20. Kalibrasi <ul><li>Menggunakan 2 tabung BD FACSCount </li></ul><ul><li>Tabung A Tabung B </li></ul><ul><li> - 1 ml sheath fluid (BD fascflow) - 3 ml sheath fluid </li></ul><ul><li> - 1 tetes unlabelled beads - masing-masing 1 tetes : </li></ul><ul><li> * unlabelled beads </li></ul><ul><li> * FITC beads </li></ul><ul><li>vortex * PE beads </li></ul><ul><li> * perCP beads </li></ul><ul><li> vortex </li></ul><ul><li>Baca dengan flow cytometer FACSCalibur </li></ul>
  21. 21. Hasil Kalibrasi
  22. 22. Terima Kasih
  23. 23. Current CD4 monitoring tests and technologies include <ul><ul><ul><li>Flow based assays </li></ul></ul></ul><ul><ul><ul><li>Becton Dickinson FACSCount </li></ul></ul></ul><ul><ul><ul><li>Guava EasyCD4 </li></ul></ul></ul><ul><ul><ul><li>Partec CyFlow </li></ul></ul></ul><ul><ul><ul><li>Beckman Coulter PointCARE </li></ul></ul></ul><ul><ul><ul><li>Manual immune bead-based assays </li></ul></ul></ul><ul><ul><ul><li>Dynal Dynabeads </li></ul></ul></ul><ul><ul><ul><li>Coulter Cytosphere </li></ul></ul></ul><ul><ul><ul><li>In the pipeline </li></ul></ul></ul><ul><ul><ul><li>LabNow microchip technology </li></ul></ul></ul><ul><ul><ul><li>SemiBio slide test (microscopy) </li></ul></ul></ul>
  24. 24. T – cell subsets T T T C H CD3 CD3 CD3 CD8 CD4
  25. 25. Foreign Invader Specific Antibody Antigen
  26. 26. Analyze Two-Color Direct Staining Incubate
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  28. 28. <ul><li>Forward Scatter—diffracted light </li></ul><ul><li>Related to cell surface area </li></ul><ul><li>Detected along axis of incident light in the forward direction </li></ul><ul><li>Side Scatter—reflected and refracted light </li></ul><ul><li>Related to cell granularity and complexity </li></ul><ul><li>Detected at 90° to the laser beam </li></ul>Right Angle Light Detector  Cell Complexity Forward Light Detector  Cell Surface Area Incident Light Source Properties of FSC and SSC
  29. 29. Side Scatter Forward Light Scatter Lysed Whole Blood 0 200 400 600 800 1000 0 200 400 600 800 1000 Lymphocytes Monocytes Neutrophils largest and most complex population smallest and least complex population
  30. 30. Limfosit subset CD3 T Llimfosit CD4 Helper T limfosit CD8 Supresor T limfosit CD19 B limfosit CD45 Lekosit CD3/CD4 Helper / Inducer T limfosit CD3/CD8 Supresor / sitotoksik limfosit CD16/CD56 NK sel CD4:CD8 Helper / supresor ratio
  31. 31. Menghitung Σ absolut CD4 <ul><li>Dengan membandingkan “cellular even” terhadap “beads even” </li></ul><ul><li>Pada BD Multiset : otomatis </li></ul><ul><li>Cara manual : </li></ul><ul><li>Absolut count of cell = Σ of events in regio Σ of beads per test </li></ul><ul><li> containing cell X </li></ul><ul><li>Σ of events in absolute test volume </li></ul><ul><li>count bead region </li></ul>
  32. 32. Aplikasi klinis flow sitometer <ul><li>Identification of minimal residual disease post chemotherapy </li></ul><ul><li>Identifying relapse of haematologic malignancies </li></ul><ul><li>Identification of antigens whose deletion correlates with increased risk for disease </li></ul><ul><ul><li>CD55 and CD59 deletion is characteristic of PNH </li></ul></ul><ul><li>Determining proliferation rates by analysing cell cycle kinetics (S phase measurement) </li></ul>

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